Method and an apparatus for dosing reagents and for their incubation and for sampling reaction mixture

ABSTRACT

A method and an apparatus for dosing reagents and for incubation and sampling of the reaction mixture, in which method the reagents are drawn into the reaction syringes in which the incubation is carried out and in which the samples are discharged from the syringes in one or more portions, the apparatus consisting of a syringe unit, reaction syringes mounted on a single frame which is easy to replace, syringes containing stopping liquid and an internal standard and mounted on a fixed outer frame, vessels for the reagents, instruments for feeding reagents and for the automatic adjustment of the pistons and for transferring absorption plates, and a thermostat. The invention is suitable for reactions and dosing purposes, particularly for macromolecular investigation of radioactive parent substances, where the sample is absorbed into a porous medium and where it is washed automatically.

. Reunanen [451 Aug. 28, 1973 METHOD AND AN APPARATUS FOR DOSING REAGENTS AND FOR THEIR INCUBATION AND FOR SAMPLING REACTION MIXTURE Matti Antero Reunanen, Kupittaank, 11-13 C 38, Turku, Finland Filed: May 10, 1971 Appl. No.: 141,511

Inventor:

Foreign Application Priority Date May 12, 1970 Finland 1323/70 US. Cl. 23/230 R, 23/253 R, 23/259, 73/4256 Int. Cl ..G01n1/14, GOln 1/16 Field of Search 23/253, 259, 230; 73/425.6

References Cited UNITED STATES PATENTS 3,650,306 3/1972 Lancaster 23/259 X Primary Examiner-Morris O. Wolk Assistant Examiner-R. E. Serwin Attorney-Sughrue, Rothwell, Mion, Zinn & Macpeak ABSTRACT A method and an apparatus for dosing reagents and for incubation and sampling of the reaction mixture, in which method the reagents are drawn into the reaction syringes in which the incubation is carried out and in which the samples are discharged from the syringes in one or more portions, the apparatus consisting of a syringe unit, reaction syringes mounted on a single frame which is easy to replace, syringes containing stopping liquid and an internal standard and mounted on a fixed outer frame, vessels for the reagents, instruments for feeding reagents and for the automatic adjustment of the pistons and'for transferring absorption plates, and a thermostat. The invention is suitable for reactions and dosing purposes, particularly for macromolecular investigation of radioactive parent substances, where the sample is absorbed into a porous medium and where it is washed automatically.

13 Claims, 3 Drawing Figures 1 METHOD AND AN APPARATUS FOR DOSING REAGENTS AND FOR THEIR INCUBATION AND FOR SAMPLING REACTION MIXTURE BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a method and an apparatus for dosing reagents and for incubation and sampling of the reaction mixture. By applying the method and apparatus of the invention, reagents can be drawn into a large number of single-use syringes from boat-like vessels, incubation can be carried out at the desired temperature and samples can be removed from syringes at predetermined intervals, or reactions can also be stopped in syringes and the entire amount analyzed at a time. The invention is particularly suitable for macromolecular investigation of radioactive parent substances, where the sample is absorbed into a porous medium and then is washed automatically (Finnish patent application 801/70, corresponding to United States Pat. Application Ser. No. 125,989, filed Mar. 19, 1971). Of course, the apparatus is suited also for other kinds of reactions and dosing purposes.

2. Description of the Prior Art Previously similar reactions have been done in test tubes, into which reagents have been pipetted one by one (in fact there are automatic pipet-devices, but with them only one reagent can be dosed at the same time), and during the incubation samples have been taken with a pipet from each tube in turn or the entire contents of the tube have been precipitated, washed and analyzed as a whole. It is particularly troublesome to sample several tubes at short intervals and confusion is easily caused. It is also troublesome to absorb the sample quantitatively from the tubes into the abovementioned automatic wash. The old methods also demand precipitation, when the sample is taken, while according to the invention, everything occurs automatically.

SUMMARY OF THE INVENTION By means of the invention with respect to the amount of work, the danger of confusion and difficulty can be diminished essentially. The composition of the reaction mixture can be controlled more closely, because the mixture is kept homogenous by mixing it. Further on it is possible to act even if there is not much oxygen. Also subsequent treatment of the sample is even. Also the automatic dosing of liquids is more exact than if it were done with a pipet (Standard Deviation in 250 pl samples being less than 1 pl). The main characteristics of dosing reagents, their incubation and sampling of reaction mixture with this new method are that the reagents are drawn into reaction syringes, in which the incubation of reagents is carried out and that samples are removed from the syringes in question in one or more portions. The reaction syringes are replaceable singleuse syringes and they have been placed in a replaceable body, which is easy to change as a whole together with syringes. According to the invention, mixing in the syringes is done with a piece of magnet which is moved by an magnet external of the syringes. A further characteristic of the method is that at the same time, when samples are taken from the reaction syringes, liquid for stopping the reaction is fed syncronously from syringes arranged for the purpose, whereby stopping liquid and the sample (reaction mixture) are fed to the same point, or that samples are absorbed into the absorption plates, into which liquid for stopping the reaction has been absorbed in advance. It is possible to act also so that the mixture of stopping liquid and sample is absorbed into the absorption plate, which consists of compact surface layers and loose material between them, into which the sample is absorbed. It is characteristic of the apparatus of the invention when carrying out the above described method that it consists of a syringe unit with a desired number of reaction syringes with pistons, which syringes are mounted on a frame, vessels for different reagents and a tank for the stopping liquid, instruments for feeding reagents and stopping liquid into the reaction syringes and respectively into the stopping liquid syringes and instruments for automatically adjusting the pistons of both types of syringes, for thermostating of the reaction syringes of the thermostat, for moving the absorption plates of the conveyor and for the absorption plates of the injector, if needed. It is also characteristic of the apparatus that the pistons of both types of syringes have a common moving mechanism, which contains a stepmotor that is drive automatically by a programmed disc or a control unit which moves the plate the desired distance; the pistons of the syringes being fixed into the plate.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Other characteristics and details of the invention are described in the following referring to-the accompanying drawings, in which FIG. I shows the apparatus of the invention in its general arrangement from above,

FIG. 2 is the apparatus seen as a partial profile and FIG. 3 is a cross sectional view of the left side at the 13th screw and the right side at the 48th syringe.

DETAILED DESCRIPTION OF THE INVENTION In the following the invention is applied to protein synthesis. However, the apparatus is suitable for all incubations as well as to dosing reagents. Samples can be taken into test tubes for further analysis. The apparatus can be operated manually by hand cranking or automatically, in which it is driven by an engine. The apparatus consists of a syringe unit 1, a thermostat 2 for thermostating the syringe unit, an absorption vessel 3 for absorbing the reaction mixture and stopping solution, a tank 4 for stopping solution, a conveyor 5 for positioning the absorption vessel under the syringe and removing it, an injector 8 with liquid tanks 7 and a cold space 6. The entire system is controlled by a control unit 9. In the syringe unit 1 there are reaction syringes 18 and syringes for stopping liquid 48. From the thermostat 2 a tube 51 leads to the syringing space and a tube 52 away from there.

Reaction syringes 18 are closed by sealings 20 and placed in the detachable body 36, which is fixed on the outer frame 35 with end bars 19, which causes the head 50 of the syringe 18 to press down closely. The pistons 17 of the syringes 18 force through the cover of the fixed body 35 and the enlargements 38 of the tops of syringe pistons 17 are supported by end bars 15 by means of a beam 16 to the moving upper part, in which the gearwheels l2 situated between panels 29 and 30 are rotated synchronically by a central gear 11 by means of screws 13 and counterparts 14. These gears make thepistons 17 move either by hand or by motor 10 in the syringes 18. The syringes 48 that contain the stopping liquid are propped against the outer frame 35 and their pistons by means of a beam 49 with screws to the moving plate 30. Under the stopping syringes there is a valve part 42, one position of which makes it possible to remove solution from the tank 4 along the tube 41 and groove (lengthwise) 39 to the syringes 9 and the other position to discharge the liquid among the sample along the groove (crosswise) 40 from each syringe separately. In the reaction syringes 18 there are plastic coated light magnets 25, which are moved by a solenoid 26 in order to mix the content so that the unit 9 gives pulsating current, which draws the magnet 28 by means of the periodically lowering iron core 27, by which the mixing magnets 25 follow. The apparatus is filled with reagents and stopping solution as follows: Tubes 21 and 22 are pushed outwards by plates 23 and 24, which are drawn down and the tubes are directed towards the reagent vessels 37. At the same time the cock 45 is turned into position, which allows the liquid to flow from the vessel 4 to the syringes 48. When an impulse is given with a bush button 43 to the control unit 9, the stepengine 10 rotates and the liquid is drawn into the syringes l8 and 48 (stopping reagents into which amino acid labelled with another radioactive isotope can be added as an internal control in the protein synthesis investigation, whereat after the wash the effi ciency of the wash phase can be estimated by burning the sample and counting the tritium and carbon-M that are gathered into separate bottles). Each reagent in turn is in this way drawn into the syringes. As the magnetic mixing is working all the time and as the last one the radioactive substance amino acid is drawn through the tubes 21 and 22 (which are capillary tubes), the starting moment of reaction is made exact and the dosing of reagents is made quantitative.

The reaction can be stopped also in the syringes, but the main function of the apparatus when taking the sample is as follows: Either by pressing the button 441 or completely automatically with a programmed disc the engine E is at a desired moment made to push the pistons of the syringes l8 and 48, whereby the sample and the stopping solution are discharged. The stopping solution is discharged through the end hole of the console 46, when the cocks 45 have been turned into position, in which the groove 40 directs the liquid from the syringe 48 to the console 46 and the reaction solution along its groove 87 to the same point, when the tubes 21 and 22 are bent with plates 23 and 24 into the corresponding grooves. The solution is absorbed pneumatically 34 into the raised vessel 3, into absorption plates 54 that are separated by plates 31, which absorption plates are in their respective absorption compartments 53 and in which the compact layers 32 enclose loose material 33 into which the sample is absorbed. The material 33 can be made of gel (eg. Sephadex), whereby the small molecules (eg. radioactive amino acid) force their way inside the gel and the big molecules (eg. protein) remain on the surface onto which they can be precipitated. By washing the soluble amino acid can be removed. The advantage of the gel is that the protein does not precipitate in great masses in which there would be inside amino acid out of reach of the washing material. After letting the sample flow the tubes 21 and 22 are again emptied and the following sample is taken in the way described above.

The reaction can be stopped also by absorbing the stopping solution into the intermediate plates in advance, at which the apparatus is simplified, but the exact use of the internal standard is not possible.

When the sample is absorbed into the plates, the conveyor 5 removes the absorption vessel 3 from underneath the syringes. They are taken into the trichloroacetic acid shower 8, which acts at the right moment by the help of the control unit 9. From there the samples come to the cold space 6, where they freeze and can be taken into the wash.

The inside frame 36 with syringes 18 can, when it is removed after treatment, he changed with another similar frame by opening the even numbered end bars 15 and 19. However, it is also possible to use the same syringes by washing them without removing them from their place by repeatedly drawing into them washing liquids and again discharging them.

What I claim is:

l. A method for dosing reagents and for incubation and sampling of a reaction mixture obtained during incubation of the reagents which comprises:

a.'drawing different reagents each from its own vessel into reaction syringes containing pistons by means of said pistons of the syringes, whereby the amount of reagent drawn into each reaction syringe is adjust ed by means of said movement of said piston of said reaction syringe, said syringes acting as reaction vessels and vessels in which the incubation of the reagents takes place;

b. mixing the reagents in said syringes;

c. removing samples from said syringes at desired intervals during incubation by means of the pistons of said syringes, and

d. bringing said samples removed from said syringes and a stopping liquid which stops the reaction into contact with each other.

2. The method of claim 1, wherein said samples are removed from said reaction syringes at the same time that said stopping liquid is passed synchronously from said syringes containing said stopping liquid, said samples and said stopping liquid being passed to the same point.

3. The method of claim 1, wherein said sample and said stopping liquid are absorbed into an absorption plate.

4. The method of claim ll, wherein said sample is absorbed into an absorption plate containing said stopping liquid absorbed therein.

5. The method of claim 1, wherein said mixing of said reagent in said reaction syringes is carried out magnetically.

6. The method of claim 1, wherein said reaction syringes are replaceable single-use syringes and said reagent vessels are changeable boat-like vessels.

7. An apparatus for dosing reagents and for incubation and sampling of a reaction mixture obtained during incubation of said reagents which comprises a reaction syringe unit containing a desired amount of reaction syringes with pistons, syringes for containing a stopping solution for stopping the reaction in said reaction mixture, vessels for said reagents and a tank for said stopping liquid, means for feeding said reagents from said reagent vessels into said reaction syringes and means for feeding said stopping liquid from the tank containing it into said stopping liquid syringes, means for automatically adjusting the movement of said pistons of said reaction syringes and said stopping liquid syringes, means for discharging the contents of said syringes into absorption plates, means for thermostating said reaction syringes during incubation, and a control unit for controlling said apparatus.

8. The apparatus of claim 7, wherein said reaction syringes are mounted on a single frame, said frame being changeable as a unit.

9. The apparatus as claimed in claim 7, including a conveyor for positioning said absorption plates under said syringe unit and removing them therefrom.

10. The apparatus as claimed in claim 7, including magnets in said reaction syringe for mixing said reaction mixture, said magnets being moved by a solenoid by means of an iron core fixed into said permanent magnet.

11. The appratus as claimed in claim 7, wherein said pistons of said reaction syringes and said stopping liquid syringes have a coupled moving means comprising a stepmotor driven automatically by a control means of said control unit, which moves said moving means the needed distance, the pistons of said syringes being fixed.

12. The apparatus as claimed in claim 7, wherein said absorption plates are separated by intermediate plates and situated in their own absorption compartments which together form a movable absorption vessel.

13. The apparatus as claimed in claim 12, wherein said absorption plate is formed of compact surface layers and loose material therebetween, said material being a gel when macromolecular synthesis is investigated.

i l i i 

2. The method of claim 1, wherein said samples are removed from said reaction syringes at the same time that said stopping liquid is passed synchronously from said syringes containing said stopping liquid, said samples and said stopping liquid being passed to the same point.
 3. The method of claim 1, wherein said sample and said stopping liquid are absorbed into an absorption plate.
 4. The method of claim 1, wherein said sample is absorbed into an absorption plate containing said stopping liquid absorbed therein.
 5. The method of claim 1, wherein said mixing of said reagent in said reaction syringes is carried out magnetically.
 6. The method of claim 1, wherein said reaction syringes are replaceable single-use syringes and said reagent vessels are changeable boat-like vessels.
 7. An apparatus for dosing reagents and for incubation and sampling of a reaction mixture obtained during incubation of said reagents which comprises a reaction syringe unit containing a desired amount of reaction syringes with pistons, syringes for containing a stopping solution for stopping the reaction in said reaction mixture, vessels for said reagents and a tank for said stopping liquid, means for feeding said reagents from said reagent vessels into said reaction syringes and means for feeding said stopping liquid from the tank containing it into said stopping liquid syringes, means for automatically adjusting the movement of said pistons of said reaction syringes and said stopping liquid syringes, means for discharging the contents of said syringes into absorption plates, means for thermostating said reaction syringes during incubation, and a control unit for controlling said apparatus.
 8. The apparatus of claim 7, wherein said reaction syringes are mounted on a single frame, said frame being changeable as a unit.
 9. The apparatus as claimed in claim 7, including a conveyor for positioning said absorption plates under said syringe unit and removing them therefrom.
 10. The apparatus as claimed in claim 7, including magnets in said reaction syringe for mixing said reaction mixture, said magnets being moved by a solenoid by means of an iron core fixed into said permanent magnet.
 11. The appratus as claimed in claim 7, wherein said pistons of said reaction syringes and said stopping liquid syringes have a coupled moving means comprising a stepmotor driven automatically by a control means of said control unit, which moves said moving means the needed distance, the pistons of said syringes being fixed.
 12. The apparatUs as claimed in claim 7, wherein said absorption plates are separated by intermediate plates and situated in their own absorption compartments which together form a movable absorption vessel.
 13. The apparatus as claimed in claim 12, wherein said absorption plate is formed of compact surface layers and loose material therebetween, said material being a gel when macromolecular synthesis is investigated. 